Spread and Molecular Characteristics of Enterobacteriaceae Carrying fosA-Like Genes from Farms in China

ABSTRACT In this study, we aimed to investigate the occurrence and molecular characteristics of fosfomycin-resistant Enterobacteriaceae isolates from pig, chicken and pigeon farms in Guangxi Province of China. A total of 200 fosfomycin-resistant strains were obtained from food animals and their surrounding environments, with the fosA, fosA3, and fosA7.5 genes being detected in 26% (52/200), 10% (20/200), and 5% (10/200), respectively. Surprisingly, three fosA7.5-producing E. coli isolates were found to be concomitant with fosA3. Most of the fosA-like-gene-positive isolates were multidrug-resistant strains and consistently possessed blaCTX-M-1/CTX-M-9, floR, and blaTEM genes. Only fosA3 was successfully transferred to the recipient strains, and the 29 fosA3-carrying transconjugants exhibited high-level resistance to fosfomycin (MIC ≥ 512 μg/mL). Multilocus sequence typing (MLST) combined with enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analyses indicated that fosA3 or fosA7.5 genes were spread by horizontal transfer as well as via clonal transmission between E. coli. We used the PCR mapping method to explore the genetic contexts of fosA-like genes, and two representative strains (fEc.1 and fEcg99-1) were fully sequenced. Six different genetic structures surrounding fosA3 were detected and one infrequent context was discovered among the conjugable fosA3-positive E. coli isolates. The five genetic environments of fosA were identified and found to be highly similar to the partial sequence of transposon Tn2921. Furthermore, whole-genome sequencing (WGS) results showed that fosA7.5 was colocalized with mcr-3, blaCMY-63, sul3, tet(A), dfrA, and a number of virulence-related factors on the same chromosomes of strains, and various insertion sequences (IS3/ISL3) were detected upstream or downstream of fosA7.5. The phylogenetic analysis revealed that both fosA7.5- and fosA3-carrying E. coli ST602 and fosA7.5-carrying E. coli ST2599 were closely related to E. coli isolates from humans, which may indicate that they pose a threat to human health. IMPORTANCE Here, we report the widespread and complex genetic environments of fosA-like genes in animal-derived strains in China. The fosA7.5 gene was identified in this study and was found to confer resistance to fosfomycin. The high prevalence of fosA-like genes in farms indicates that food animals serve as a potential reservoir for the resistance genes. This study also discovered that fosfomycin resistance genes were always associated with mobile elements, which would accelerate the transmission of fosA-like genes in strains. Importantly, E. coli ST602 and ST2599 carrying fosA3 or fosA7.5 from food animals had high similarity to E. coli isolates from humans, suggesting that fosA-like genes can be transmitted to humans through the food chain, thus posing a serious threat to public health. Therefore, the prevalence of fosA-like genes isolated from animals should be further monitored.

6. L47-49, explaining that this work is important because it is important to complete the work is a circular argument. Why is completing the work important, what will that work inform us of? 7. L70, By America, do you mean the USA, or North America or South America or both? If the whole hemisphere, please refer to it as, "the Americas" 8. L84, it is not co-localized, please rephrase. 9. L128, This is not correct, they do not coexist, rather the are both in the same isolate. 10. L131, Table 3? Where are Table 1 and 2? Please reorganize the order of the tables and figures to follow the text. 11. L391 Awkward sentence, please rewrite it to make it clear to the reader what you mean. 12. L397, Awkward sentence, please rewrite. 13. L412, "verified in THIS study"? I think you mean it should be verified in a subsequent study.
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Thank you for submitting your paper to Microbiology Spectrum. While we are willing to consider a revised version of this paper at Spectrum, it would be in your best interest to improve the writing. I recommend that you ask a colleague of yours who is a native English speaker to read and provide you some feedback on the writing. You are also welcome to use one of the services here: https://journals.asm.org/content/language-editing-services While we are willing to consider a revised version of this paper at Spectrum, it would be in your best interest to improve the writing. I recommend that you ask a colleague of yours who is a native English speaker to read and provide you some feedback on the writing. You are also welcome to use one of the services here: https://journals.asm.org/content/language-editing-services In this study, Zhang et al. identified fosA-like genes in Enterobacteriaceae and the genetic context of such elements. The study shows that the identified fosA-like genes are frequently associated with mobile elements, which may suggest that these genes are highly transferable, allowing for their dissemination within microbial populations. Additionally, many of the isolates containing fosA-like elements also contained other antibiotic resistance genes. I recommend rewriting sections of the manuscript and have provided some examples below. I have a few suggestions for the authors to consider: 1. Move "(MDR)" on line 54 to after "multidrug-resistant" and before "Gram-negative bacteria" 2. Move "such as extended-spectrum--lactamase (EBSL)-producing Enterobacteriaceae and carbapenem-resistent Enterobacteriaceae (CRE)" after "has resulted in an increase in the number of bacterial infections" and before "while limiting the availability of antibiotics". 3. More information on fosA and fosA-like genes should be included in the introduction (i.e., clarification on which type of bacteria carry fosA genes, what the differences are between fosA and fosA-like genes). 4. The numbering of the tables included in the manuscript is off. The first table referenced in the manuscript should be titled " Table 1". 5. Please define "ARGs" on line 119. 6. Please add the antibiotics that the genes listed in lines 118-129 confer resistance to in parentheses. 7. Please define PLSDB on line 232. 8. Please differentiate between supplemental tables and supplemental datasets. 9. Please elaborate more on the correlation between bacterial resistance and virulence mentioned in the discussion. 10. Were any quality checks performed on the assembled sequences (i.e., those isolates containing fosA7.5)? 6. A revision of the use of English language is highly recommended.

Minor issues
1. Line 143. Enterobacteriaceae are intrinsically resistant to erythromycin. The evaluation of the antimicrobial activity of erythromycin adds limited information to the manuscript.
3. Line 154. The mechanism of resistance to meropenem should be investigated, since carbapenem resistance is an important clinical issue.
6. Please report all species and genes in italics.

2.
Move "such as extended-spectrum--lactamase (EBSL)-producing Enterobacteriaceae and carbapenem-resistent Enterobacteriaceae (CRE)" after "has resulted in an increase in the number of bacterial infections" and before "while limiting the availability of antibiotics".
3. More information on fosA and fosA-like genes should be included in the introduction (i.e., clarification on which type of bacteria carry fosA genes, what the differences are between fosA and fosA-like genes).
4. The numbering of the tables included in the manuscript is off. The first table referenced in the manuscript should be titled "

Alterations in glpT and uhpT genes should be checked in the strains subjected
to Whole Genome sequencing in order to exclude the presence of other mechanisms of resistance to fosfomycin.
As suggested by the reviewer, the chromosome-mediated fosfomycin resistance mechanism of the two strains sequenced by the whole genome was analyzed, and it was found that the uhpT gene sequences of strains fECg99.1 and fEC.1 were identical to the reference strain K12. There were two amino acids were changed in GlpT of fEC.1, and some sequences were missing. While the target enzyme MurA was not changed in the two strains. However, in strain fEC.1, fosA3 is located on a conjugative IncFII plasmid. We verified by conjugation test that after obtaining fosA3-carrying IncFII plasmid, the MICs of fosfomycin to the recipient bacteria was ≥ 512μg/mL. In strain fECg99.1, we tested the function of the fosA7.5 gene, and the results showed that the fosA7.5 gene identified in this study can confer high-level resistance to fosfomycin (MIC>128 μg/mL).

Lines 455-458. Fosfomycin should be tested with agar dilution method on
Mueller Hinton agar as recommended by ISO 20776-1:2019, CLSI and

EUCAST.
As suggested by the reviewer, the content of "Antimicrobial susceptibility testing" has been modified, and the modifications can be seen on lines 479-482.

Please add in the Discussion section a possible explanation of the high prevalence of acquired fosfomycin resistance genes in the veterinary setting and
which measures should be taken in order to limit their spread.
As suggested by the reviewer, this aspect has been added to the "discussion".
Modifications can be seen on lines 380-390. In addition, according to the suggestions of reviewers, the "discussion" section of the manuscript has been modified to provide an in-depth discussion of the findings, and the modifications can be seen on lines 351-443.

Lines 312-350. ERIC PCR and MLST analysis sections should be combined and results reported together.
As suggested by the reviewer, the ERIC PCR and MLST analysis sections have been combined, and the results also reported together. Modifications can be seen on lines 203-234.

A revision of the use of English language is highly recommended.
As suggested by the reviewer, the use of English language throughout the article has been revised.

Minor issues
1. Line 143. Enterobacteriaceae are intrinsically resistant to erythromycin. The evaluation of the antimicrobial activity of erythromycin adds limited information to the manuscript.
As suggested by the reviewer, the evaluation of the antibacterial activity of erythromycin and rifampicin in "Detection of Antimicrobial Resistance Patterns" has been removed, and the analysis of the results has been revised in detail. Modifications can be seen on lines 155-172.

Line 90. "FOSA" protein should be written as "FosA".
As suggested by the reviewer, the "FOS" that appears throughout the article has been changed to "Fos".
3. Line 154. The mechanism of resistance to meropenem should be investigated, since carbapenem resistance is an important clinical issue.
As suggested by the reviewer, the resistance mechanism of these strains to meropenem has been analyzed, and the results showed that meropenem-resistant strains carried bla NDM resistance gene. Modifications can be seen on lines 129-131 and Table 2.

Line 158. Please rephrase.
As suggested by the reviewer, the sentence on line 158 has been restated and changed to "Except for one strain that was only resistant to two antibiotics (fosfomycin and florfenicol), all 81 strains carried fosA-like genes were multidrug-resistant strains (resistant to at least 3 classes agents)". Modifications can be seen on lines168-170.

Lines 369-370. 9 RmtB-producing isolate.
As suggested by the reviewer, the sentence on lines 369-370 has been modified, and the modifications can be seen on lines 385-386.

Please report all species and genes in italics.
As suggested by the reviewer, the all species and genes in the text have been changed to italic status.
As suggested by the reviewer, the content in the "Introduction" has been revised, and the modifications can be seen on lines 58-61.

Move "such as extended-spectrum--lactamase (EBSL)-producing
Enterobacteriaceae and carbapenem-resistent Enterobacteriaceae (CRE)" after "has resulted in an increase in the number of bacterial infections" and before "while limiting the availability of antibiotics".
As suggested by the reviewer, the content in the "Introduction" has been revised, and the modifications can be seen on lines 58-61.
3. More information on fosA and fosA-like genes should be included in the introduction (i.e., clarification on which type of bacteria carry fosA genes, what the differences are between fosA and fosA-like genes).
As suggested by the reviewer, the content in the "Introduction" has been revised, which on lines 73 and 88-90. The "fosA-like genes" represent all genes that have been identified to encode FosA enzymes, including fosA1-fosA10 et al. The "fosA gene" that appears in the manuscript represents the "fosA1 gene". As the researchers used to describe the gene as "fosA" in previous research reports, this paper also describes "fosA1" as "fosA".
4. The numbering of the tables included in the manuscript is off. The first table referenced in the manuscript should be titled " Table 1".
As suggested by the reviewer, the tables and figures appearing in the manuscript have been presented in order.

Please define "ARGs" on line 119.
As suggested by the reviewer, we have defined "ARGs" as "antibiotic resistance genes (ARGs)" on line 116.

Please add the antibiotics that the genes listed in lines 118-129 confer resistance to in parentheses.
As suggested by the reviewer, we have added the antibiotics that the genes confer Since the BLAST method was used to screen out plasmids with higher similarity, the method of PLSDB was deleted. The modifications can be seen on lines 272-274.

Please differentiate between supplemental tables and supplemental datasets.
As suggested by the reviewer, the Supplementary Tables and Supplementary Figures have been added to the "Supplementary Materials".
9. Please elaborate more on the correlation between bacterial resistance and virulence mentioned in the discussion.
Since the results of this study showed that there were multiple virulence-related factors around fosA7.5, but the relationship between the resistance gene and virulence was not confirmed, this section was deleted from the "Discussion".
The whole genome sequences, which we obtained were quality-checked.

Reviewer 3 1. English usage.
As suggested by the reviewer, the use of English language throughout the article has been revised.

L.21-25, what does concomitant mean in this sentence?
"Concomitant with" in the text means that three fosA7.5-carrying E. coli strains were also identified to carry the fosA3 gene.
As suggested by the reviewer, "successfully obtained" has been changed to "was successfully transferred" on line 27.

L.42, connected is the wrong word.
As suggested by the reviewer, the "Importance" content in the "Abstract" has been revised. The modifications can be seen on lines 44-54. As suggested by the reviewer, the incorrect expression in the "importance" section of the "Abstract" has been revised. The modifications can be seen on lines 44-54.

L47-49, explaining that this work is important because it is important to
complete the work is a circular argument. Why is completing the work important, what will that work inform us of?
As suggested by the reviewer, the importance of this work has been described in "importance". The work indicated that the food animals served as a potential reservoir for the resistance genes, and the mobile elements would accelerate the transmission of fosA-like genes in strains. In addition, the E. coli carrying fosA-like genes from food animals had a high similarity with E. coli isolates from humans, suggesting that fosA-like genes can be transmitted to humans through the food chain, thus posing a serious threat to public health. The modifications can be seen on lines 46-53.
7. L70, By America, do you mean the USA, or North America or South America or both? If the whole hemisphere, please refer to it as, "the Americas".
As suggested by the reviewer, "America" in the manuscript has been changed to "the Americas", and is on line 69.

L84, it is not co-localized, please rephrase.
As suggested by the reviewer, this part of the "Introduction" has been re-described, and the modifications can be seen on lines 77-80.
9. L128, this is not correct, they do not coexist, rather the are both in the same isolate.
As suggested by the reviewer, this part of the "Identification of fosfomycin-resistant determinants and the coexistent-resistant genes" has been re-described, and the modifications can be seen on lines 126-128.
10. L131, Table 3? Where are Table 1 and 2? Please reorganize the order of the tables and figures to follow the text.
As suggested by the reviewer, the tables and figures appearing in the manuscript have been presented in order.
11. L391 Awkward sentence, please rewrite it to make it clear to the reader what you mean.
As suggested by the reviewer, this sentence has been revised, and the modifications can be seen on lines 412-413.

L397, Awkward sentence, please rewrite.
As suggested by the reviewer, this sentence has been revised, and the modifications can be seen on lines 418-421.

L412, "verified in THIS study"? I think you mean it should be verified in a subsequent study.
As suggested by the reviewer, the whole part of the "discussion has been revised".
In addition to this, each corresponding figure has been modified according to the revision of the content in the manuscript.